Assessment of cervical cells

ABSTRACT

A method of determining abnormality in a tissue sample containing cells of the cervix the method comprising determining binding of specific binding substances to the sample and comparing the binding with the pattern of binding of said specific binding substances to a normal cervical cell sample. The specific binding substances may be antibodies. Hybridomas which produce suitable antibodies have been deposited at the European Collection of Animal Cell Cultures (ECACC), Centre for Applied Microbiology &amp; Research, Salisbury, Wiltshire SP4 0JG, United Kingdom on Feb. 6, 1995 under the accession numbers ECACC 95020718, 95020716, 95020720, 95020717 and 95020719.

[0001] The present invention relates to assessment of cells in a sampleof tissue containing cells of the cervix. More particularly, it relatesto evaluation of the state of cells of the cervix, discriminatingbetween normality and some deviation from normality, and is generallyfor use in screening women to detect those whose cervical cells areabnormal. Samples found to be abnormal may be examined in more detailand the condition of cells in the cervix investigated further.Identification of a malignant or pre-malignant condition is typicallyfollowed by appropriate treatment following more extensive diagnosticprocedures.

[0002] Cancer of the cervix is the second most common cancer in women.The current method of detection is the Papanicolau or PAP test, whichuses conventional cytological dyes to stain cells in a smear sample,enabling the visual detection of cellular nuclei and cytoplasm with amicroscope. Trained personnel make semi-subjective assessments of thenormality or otherwise of the cells examined. The PAP test, thoughuniversally accepted, is labour-intensive and prone to human error, asevidenced by a number of recent well-publicised scares casting doubt onthe accuracy of assessments made by a few of those people who spendtheir entire day looking at smear samples.

[0003] An alternative or more objective way of assessing the state ofcells in cervical cell samples would be useful and advantageous. Benefitwould be obtained by the removal or at least amelioration of problemsarising from the need for subjective, visual evaluation.

[0004] It is known that cells within tissues of the mammalian bodyexpress cellular markers that are either unique or partially restrictedto particular cell populations. Different cell populations may bedistinguished by virtue of their individual cell markers. Thus, a cellmay be shown to belong to a particular cell population (eg lymphoidcells) because of its expression of markers defining that population.

[0005] These cell markers may be various kinds of molecules, includingproteins, lipids, carbohydrates and combinations of these, such asglycoproteins, glycolipids and lipoproteins.

[0006] It is possible to detect cell markers using binding molecules,such as antibodies, with the requisite specificity. Such bindingmolecules can be used in qualitative or quantitative detection of cellswhich bear particular markers and are thus included in a particular cellpopulation.

[0007] Holmes et al have described previously a monoclonal antibody ableto bind specifically to an antigen on normal hepatocytes within theliver but not able to bind cells other than hepatocytes. No bindingcould be detected on a number of transplanted and primarydimethylaminoazobenzene-induced hepatomas nor on liver cells frompatients with a variety of liver diseases. (See: Tumour ProgressionMarkers—Proceedings of the Sixth Meeting of the European Association forCancer Research, Budapest Oct. 12-15, 1981, 471-481 (1982); Liver(1983), 3: 295-302; Int. J. Cancer (1982), 29: 559-565; Cancer Research(1984), 44: 1611-1624.)

[0008] The present invention is founded in the realisation that it ispossible to recognise a pattern of surface antigens on cells of thecervix which represents normality, such that a deviation from adetermined and noted pattern of normality can be perceived.Conveniently, antibodies or other specific binding molecules may be usedin the qualitative and/or quantitative detection of marker antigens onthe cells, enabling increased or reduced expression or loss of one ormore of the markers to be correlated with a disease (or pre-disease)state. In screening, this enables samples with some deviation fromnormality to be identified and examined further, ie suspect samples arehighlighted for further examination by suitably qualified personnel. Ifabnormality in a particular sample is serious or potentially serious,appropriate steps may be taken to examine and then perhaps treat thewoman from whom the sample was obtained. Diagnosis and decisions on theneed for and nature of treatment remain the domain of clinicians.

[0009] For operation of the present invention it is not necessary forthe marker antigens actually employed in the test to be identified.Ultimately, what is important is that a panel of antibodies or otherbinding molecules is identified as able to bind cervical cells with apattern which is associated with the cervix being normal, and thatdeviation of binding of those binding molecules from the pattern ofnormality is correlated with the onset of pathological conditions. Theonset of pathology may then be identified in samples containing cervicalcells by some deviation from the established pattern of normality forthose particular binding molecules.

[0010] This is exemplified by data included herein relating to fivemonoclonal antibodies able to bind to various cells of the cervix. Thepattern of binding of these antibodies to an abnormal tissue samplecontaining cervical cells is different from the binding to normalcervical samples enabling identification of abnormality. Clearly, otherspecific binding molecules may be employed successfully in aspects ofthe present invention as long as the requirements set out in thepreceding paragraph are satisfied. Such other molecules may bind theantigens bound by the exemplified antibodies, at the same or differentepitopes. Indeed they may bind different antigens altogether.

[0011] According to a first aspect of the present invention there isprovided a method of determining abnormality in a tissue samplecontaining cells of the cervix, the method comprising determiningbinding of antibodies to the sample and comparing the binding with thepattern of binding of said antibodies to a normal cervical cell sample.The pattern of binding of the antibodies to normal cervical cells may,and generally will, be established prior to performance of the method.

[0012] The reactivities of antibodies on normal and test samples may bedetermined by any appropriate means. Tagging with individual reportermolecules is one possibility. The reporter molecules may directly orindirectly generate detectable, and preferably measurable, signals. Thelinkage of reporter molecules may be directly or indirectly, covalently,eg via a peptide bond or non-covalently. Linkage via a peptide bond maybe as a result of recombinant expression of a gene fusion encodingantibody and reporter molecule.

[0013] One favoured mode is by covalent linkage of each antibody with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

[0014] Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

[0015] The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

[0016] The signals generated by individual antibody-reporter conjugatesmay be used to derive quantifiable absolute or relative data of therelevant antibody binding in normal and test samples. In addition, ageneral nuclear stain such as propidium iodide may be used to enumeratethe total cell population in a sampled smear, allowing the provision ofquantitative ratios of individual cell populations relative to the totalcells in a smear, at least where individual antibody reactivitiescorrelate with particular cervical cell populations.

[0017] An actual expansion or reduction in the absolute numbers of aparticular cell population is not a necessary pre-requisite for thepurposes of this invention. Any change in the detection of antibodybinding (and by implication change in the phenotypic cell markers) thatis detectable, and preferably quantifiable, relative to establishedparameters of normality, is of relevance.

[0018] In addition, non-epithelial cells such as leukocytes are known toinfiltrate cervical tissue as a consequence of pathological conditions.These may be enumerated by use of readily available monoclonalantibodies against pan-leukocytic markers, thus providing a furtherlevel of analysis.

[0019] Antibodies which are specific for a target of interest may beobtained using techniques which are standard in the art. Methods ofproducing antibodies include immunising a mammal (eg mouse, rat, rabbit,horse, goat, sheep or monkey) with the protein or a fragment thereof ora cell or virus which expresses the protein or fragment. Immunisationwith DNA encoding the target polypeptide is also possible. Antibodiesmay be obtained from immunised animals using any of a variety oftechniques known in the art, and screened, preferably using binding ofantibody to antigen of interest. For instance, Western blottingtechniques or immunoprecipitation may be used (Armitage et al, 1992,Nature 357: 80-82).

[0020] The production of monoclonal antibodies is well established inthe art. Monoclonal antibodies can be subjected to the techniques ofrecombinant DNA technology to produce other antibodies or chimericmolecules which retain the specificity of the original antibody. Suchtechniques may involve introducing DNA encoding the immunoglobulinvariable region, or the complementarity determining regions (CDRs), ofan antibody to the constant regions, or constant regions plus frameworkregions, of a different immunoglobulin. See, for instance, EP184187A, GB2188638A or EP-A-0239400. A hybridoma producing a monoclonal antibodymay be subject to genetic mutation or other changes, which may or maynot alter the binding specificity of antibodies produced.

[0021] As an alternative or supplement to immunising a mammal with apeptide, an antibody specific for a target may be obtained from arecombinantly produced library of expressed immunoglobulin variabledomains, eg using lambda bacteriophage or filamentous bacteriophagewhich display functional immunoglobulin binding domains on theirsurfaces; for instance see WO92/01047. The library may be naive, that isconstructed from sequences obtained from an organism which has not beenimmunised with the target or may be one constructed using sequencesobtained from an organism which has been exposed to the antigen ofinterest (or a fragment thereof).

[0022] Antibodies may be modified in a number of ways. Indeed the term“antibody” should be construed as covering any specific bindingsubstance having a binding domain with the required specificity. Thusthis covers antibody fragments, derivatives, functional equivalents andhomologues of antibodies, including any polypeptide comprising animmunoglobulin binding domain, whether natural or synthetic. Chimaericmolecules comprising an immunoglobulin binding domain, or equivalent,fused to another polypeptide are therefore included. Cloning andexpression of chimaeric antibodies are described in EP-A-0120694 andEP-A-0125023.

[0023] It has been shown that the function of binding antigens can beperformed by fragments of a whole antibody. Example binding fragmentsare (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii)the Fd fragment consisting of the VH and CH1 domains; (iii) the Fvfragment consisting of the VL and VH domains of a single antibody; (iv)the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) whichconsists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments (vii)single chain Fv molecules (scFv), wherein a VH domain and a VL domainare linked by a peptide linker which allows the two domains to associateto form an antigen binding site (Bird et al, Science, 242, 423-426,1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecificsingle chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”,multivalent or multispecific fragments constructed by gene fusion(WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448,1993).

[0024] Hybridomas able to produce monoclonal antibodies of use in thepresent invention have been deposited and represent individual aspectsof the present invention, as do the monoclonal antibodies themselves.Thus, the present invention provides individually each of the hybridomasdeposited as ECACC 95020718, 95020716, 95020720, 95020717 and 95020719,and mutants, derivatives and descendants of each of these hybridomas,whether or not able to produce antibody of the same or alteredspecificity.

[0025] The present invention also encompasses use of the hybridomas andantibodies obtainable therefrom in the obtention of other antibodies ofuse in the assessment of the state/condition of cervical cells in atissue sample, ie able to bind to an antigen found on the surface of oneor more cell types of the cervix. Such use may involve isolation of theantigen bound by any of the antibodies obtainable from the depositedhybridomas and use of the antigen in raising further antibodies, eg byimmunisation and/or screening of ‘phage libraries, as discussed above.The antigen may, for example, be isolated by immuno-precipitation fromcervical cell extracts and then used as an immunogen eg for generationof further monoclonal antibodies, or in screening a ‘phage library, asappropriate.

[0026] Additionally, nucleic acid encoding one of the antibodies may beisolated from any of the hybridomas and used in a recombinant expressionsystem to produce whole antibody, an antibody fragment or chimaeras ofantibody/antibody fragment fused to another polypeptide (eg label suchas a peptide tag or enzyme). As mentioned above, cloning and expressionof chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023.

[0027] Recombinant expression of polypeptides, including antibodies andantibody fragments, is well-known in the art.

[0028] Systems for cloning and expression of a polypeptide in a varietyof different host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells and many others. A common, preferred bacterial host is E. coli.

[0029] Suitable vectors can be chosen or constructed, containingappropriate regulatory sequences, including promoter sequences,terminator fragments, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate. For further detailssee, for example, Molecular Cloning: a Laboratory Manual: 2nd edition,Sambrook et al, 1989, Cold Spring Harbor Laboratory Press.Transformation procedures depend on the host used, but are well known.

[0030] Thus, the present invention extends to any antibody or antibodyfragment able to bind an antigen to which any (ie one or more) of theantibodies obtainable from the deposited hybridomas is able to bind.Ability to bind the same antigen may be assessed, for example in abinding inhibition assay or in a band-shift assay on an electrophoreticgel.

[0031] The present invention also provides in a further aspect the useof any antibody or antibody fragment of the invention, including thoseobtainable from any of the deposited hybridomas, in assessment of thenature or condition of cells of the cervix in a tissue sample, asdisclosed.

[0032] Further aspects and embodiments of the present invention will beapparent to those skilled in the art.

[0033] Before illustrating embodiments of the present invention in moredetail by way of example, it is helpful to understand the organisationof the cervix and the relationships between the different cell-typesfound there.

[0034] Cell Populations of the Cervix in Normal and PathologicalConditions

[0035] The cervix is essentially composed of two distinct cell types:the squamous epithelium and the columnar epithelium, each of which islocated in an anatomically distinct region of the tissue. The squamousepithelium is located at the exterior aspect (the exocervix) of thecervical opening (the cervical os), while the columnar epitheliumextends into the endocervical canal (the endocervix). These two distinctepithelial cell types come into contact in the vicinity of the cervicalos, at the squamo-columnar junction. The squamo-columnar junction is ofclinical importance as it is the region where the majority ofmalignancies arise. For diagnostic validity, a cervical smear samplemust include cells from this region. In order to ensure that this hasbeen achieved, a smear must contain columnar as well as squamousepithelial cells.

[0036] Columnar cells are the source of cervical mucous. They aregenerally arranged as a single cell layer lining the endocervix, whichis thrown into deep folds to form the cervical glands. A smallproportion (5%) of cervical tumours are derived from columnar cells i.e.the adenocarcinomas.

[0037] In some cervices, a cuboidal cell layer, the so-called reservecell population, is disposed beneath the columnar cells. The role ofreserve cells is unclear, but believed by many investigators to giverise to columnar epithelium.

[0038] In contrast to the columnar epithelium, the squamous epithelium,from which most (95%) cervical tumours arise, is a multilayered dynamicstem cell system under constant renewal.

[0039] The stem cell compartment itself is located adjacent to thebasement membrane within the basal cell layer. Stem cell division givesrise to parabasal, intermediate, and superficial cell derivatives. Theseare conventionally defined in terms of both their characteristicmorphology and location within the squamous epithelium. The transitionfrom basal cells located in the deepest layer of the squamousepithelium, to superficial cells at its surface is associated withprogressive differentiation and a loss of proliferation untilsuperficial squamous epithelial cells at the cervical surface areterminally differentiated.

[0040] The transition zone (TZ), adjacent to thesquamo-columnar-junction is of clinical importance, as it includes aregion of metaplastic squamous-epithelium. This is generated at puberty,in response to the acidic environment of the vagina. The data presentedherein quantify the proportion of samples where the TZ is present.

EXEMPLIFICATION OF EMBODIMENTS OF THE PRESENT INVENTION BY WAY OFILLUSTRATION AND NOT LIMITATION

[0041] Abbreviations

[0042] ATCC—American Type Culture Collection; CIN—Cervicalintra-epithelial neoplasia; CHAPS(3-[Cholamidopropyl)-dimethylammonio]-1-propanesulfonate); CD—ClusterDesignation nomenclature of established cell markers; DAB—Di-aminobenzidine; ECACC—European Collection of Animal Cell Cultures;EDTA—Ethylene di-amine tetra acetic acid; HLA—Human Leukocyte Antigen;HRPO—Horse radish peroxidase; Ig—Immunoglobulin; kDA—Kilo dalton;Mab(s)—Monoclonal antibody(ies); Mwt—Molecular weight; NS1—Non-secretor1; PAGE—Polyacrylamide gel electrophoresis; PBS—Phosphate bufferedsaline; PAP—Papanicolau test; SDS—Sodium dodecyl sulphate; TBS—Trisbuffered saline; TZ—Transition zone; W/V—Weight/volume ratio.

[0043] Cell Markers

[0044] The work disclosed herein shows that under normal conditions,cell surface markers may be gained or lost as cells within a givenlineage proceed along their differentiation pathway. Therefore,monoclonal antibodies or other binding molecules exhibiting specificreactivities against these markers provide a means of monitoring thenormal progression of cells along their differentiation pathway. Inpathological conditions, the normal expansion or loss of these markersmay be perturbed. Consequently, an expansion or loss of a cell surfacemarker is detectable as a particular cell population proliferates orbecomes arrested at a discrete stage in its differentiation pathway.

[0045] The antibody reactivities described herein illustrate thesituation where an overlapping continuum of reactivities is observedthroughout normal squamous epithelial cell differentiation in the humancervix.

[0046] The designated nomenclature of the five monoclonal antibodiesexemplified herein is:

[0047] 1. CV3.6B5/F3/C2, hybridoma deposited as ECACC 95020718

[0048] 2. 2C7/B4/D6., hybridoma deposited as ECACC 95020716

[0049] 3. CV5.9G5.C6, hybridoma deposited as ECACC 95020720

[0050] 4. HG3/E11/C4, hybridoma deposited as ECACC 95020717

[0051] 5. BC4/E7/E5, hybridoma deposited as ECACC 95020719

[0052] Generally herein, these antibodies are referred to in theirabbreviated forms. The hybridomas were deposited at European Collectionof Animal Cell Cultures (ECACC), Centre for Applied Microbiology &Research, Salisbury, Wiltshire SP4 0JG, United Kingdom on Feb. 6, 1995.

[0053] Thus: Basal cells are 6B5+ BC4− 9G5− HG3− 2C7− Parabasal cellsare 6B5+ BC4+ 9G5− HG3− 2C7− Intermediate cells are 6B5− BC4+ 9G5+ HG3+2C7− Superficial squames are 6B5− BC4− 9G5+ HG3+ 2C7− Columnar cells are6B5+ BC4− 9G5− HG3− 2C7+.

[0054] These characteristic antibody reactivity profiles reproduciblydefine discrete stages in the differentiation of squamous epithelialcells. Additionally, they readily allow columnar epithelial cells to bedistinguished from squamous epithelial cells.

[0055] Brief Description of Results

[0056] Immunoprecipitation of the 6B5 target protein from the cellsurface of a cervical carcinoma cell line and from membrane preparationsof cervical squamous epithelium:

[0057] (a) Cell surface proteins on the cervical carcinoma cell lineC4II (Auersperg 1969. J. Natl. Cancer Inst. USA 43 151-173) wereradioiodinated by the lactoperoxidase method (Marchalonis 1969. BiochemJ. 113 299-305). Cells were solubilised with the detergent CHAPS(3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate).Immunoprecipitations were carried out with test Mab against equalvolumes of lysate (Houlihan et. al. 1992 J. Immunol. 149 668-675). Theresults show that Mab 6B5 detects a cell surface dimeric protein withcomponents of approx. 181 and 184 kDa in a cervical epithelial cellline.

[0058] (b) Standard membrane extracts were prepared from sheets ofcervical squamous epithelium (Evans 1979. Laboratory techniques inbiochemistry and molecular biology, Eds. Work and Work 7 1-266,Elsevier). Membranes were radioiodinated using Iodobeads (Markwell 1982.Anlyt. Biochem. 125 427-432) and solubilised in CHAPS.Immunoprecipitations were carried out as described above. The resultsshow that a similar form of this protein is also present in amembrane-associated fraction of cervical squamous epithelium shown in(a).

[0059] Immunoprecipitated material was analysed by SDS-PAGE underreducing conditions and autoradiography (Laemmli 1970. Nature 227133-681).

[0060] Affinity Isolation of the 6B5 Target from C4II Cells

[0061] Affinity chromatography was performed as described by Houlihan etal. 1992 (J. Immunol. 149 668-675). Purified 6B5 antibody was conjugatedto protein G-sepharose and cross-linked with dimethyl pimelimidate.2×10⁸ C4II cells were solubilised in the detergent CHAPS. The lysate waspassed through a preclearing column containing mouse Ig-sepharose andprotein A sepharose (Sigma) and then through the 6B5 antibody column.Both columns were washed with 10 bed volumes of lysis buffer and theneluted with 50 mM triethylamine, pH 11.5. The eluate was neutralisedwith 2 M glycine pH 2.0, concentrated by microultrafiltration andanalysed by SDS-PAGE under reducing conditions and coomassie bluestaining.

[0062] A single 180 kDa product was specifically isolated by the 6B5column and detected in reduced eluates. This component was not observedin the eluate representing the negative control—mouse Ig/protein Asepharose column. Immunoglobulin H-chain which had leached duringelution from the negative control column was present, however, in themouse Ig/protein A sepharose eluate.

[0063] Immunoblotting of Detergent-Solubilised Endocervical Materialusing Antibody 2C7

[0064] Endocervical tissue was minced in Hanks buffered saline andproteins were solubilised in CHAPS(3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate) buffer. Thedetergent soluble material was concentrated by microultrafiltrationusing a 30 kD cut-off filter. Proteins were electrophoreticallyseparated on 1% agarose gels in Laemmli sample buffer under reducingconditions. Proteins were transferred to microporous membranes forimmunoblotting. Identical strips were probed with Mab 2C7 and tissueculture supernatant as a negative control. Parallel strips were stainedwith periodic acid-Schiffs reagent which stains glycoproteins, andcoomassie blue as a general protein stain (Methods were based on thoseof Morales et. al. 1993 Human Reproduction 8 78-83). -ve controlimmunoblot employed tissue culture supernatant.

[0065] Mab 2C7 specifically detects components migrating in the highm.wt. range that correspond to material stained by periodicacid-Schiff's reagent. The large m.wt (>500 kDa) of this material anddetection by periodic acid-Schiff's reagent is consistent with thepresence of mucins. The Mab showed no reactivity with any proteins inthe significantly lower m.wt. material stained by coomassie blue.

[0066] Immunoblotting of Detergent-Solublised Cervical Epithelium usingMab 9G5

[0067] Squamous epithelial sheets were isolated from normal cervicesusing the enzyme Dispase (Boehringer). Sheets were solubilised in thedetergent CHAPS(3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate).Cytokeratin-enriched extracts were prepared from the CHAPS-insolublematerial by the method of Franke et al. 1981 (Exp. Cell Res. 131209-213). Both detergent (D) and cytokeratin (C) extracts wereelectrophoretically-separated by SDS-PAGE under reducing conditions(Laemmli 1970 Nature 227 133-681) and proteins were transferred tomicroporous membranes for immunoblotting (Towbin et al. 1979. Proc.Natl. Acad Sci USA 76 4350-4354).

[0068] Mab 9G5 detects a single 40 kDa product in detergent-solubilisedextracts but not in cytokeratin extracts of cervical squamousepithelium. A pan-reactive cytokeratin Mab AH3 (J. M. Houlihan Ph.D.thesis University of Bristol 1993) was used as a control; keratins aredetected in the cytokeratin extract.

[0069] Affinity Chromatographic Isolation of the 9G5 Target Protein fromHuman Amnion Cells

[0070] Affinity chromatography was performed as described by Houlihan etal. 1992 (J. Immunol. 149 668-675). Purified Mab 9G5 was conjugated toprotein G-sepharose and cross-linked with dimethyl pimelimidate. 2×10⁸amnion cells were isolated from the term placental membranes byincubation in trypsin followed by collagenase/hyaluronidase by themethod of Holmes et al. 1990. (J. Immunol. 144 3099-3015) andsolubilised in the detergent TX-100. The lysate was passed throughpreclearing columns, comprising mouse Ig and protein A sepharose andthen the Mab 9G5 column. The Mab 9G5 column was eluted with 50 mMtriethylamine, pH 11.5. The eluate was concentrated bymicroultrafiltration and analysed by SDS-PAGE under non-reducingconditions and coomassie blue staining.

[0071] A single 40 kDa product was isolated from amnion cell lysates bythe Mab 9G5 affinity column. A fraction of this eluate was examined byimmunoblotting with the 9G5 Mab and an irrelvant IgG1 Mab as a negativecontrol. The 9G5 reacted with the 40 kDa product. The strip probed withthe negative control Mab was blank.

[0072] Immunoblotting of Detergent-Solubilised Cervical Epithelium usingMab HG3

[0073] Squamous epithelial sheets were isolated from normal cervicesusing the enzyme Dispase (Boehringer). Sheets were solubilised in thedetergent CHAPS(3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate). Equalamounts of the CHAPS-soluble material were electrophoretically-separatedby SDS-PAGE (Laemmli 1970. Nature 227 133-681) under both non-reducingand reducing conditions. Proteins were transferred to microporousmembranes for immunoblotting (Towbin et al. 1979. Proc. Natl. Acad SciUSA 76 4350-4354). Parallel strips tested with Mab HG3; tissue culturesupernatant was used as a negative control.

[0074] The negative control blot was blank.

[0075] (NR—non-reduced; R—Reduced)

[0076] Mab HG3 detects a product of approx. 180 kDa, under bothnon-reducing and reducing conditions, in detergent soluble cervicalsquamous epithelium.

[0077] Immunoblotting of Detergent-Soluble and Detergent-InsolubleCervical Epithelium using Mab BC4

[0078] Squamous epithelial sheets were isolated from normal cervicesusing the enzyme Dispase (Boehringer). Sheets were solubilised in thedetergent CHAPS(3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate).CHAPS-soluble and CHAPS-insoluble material was adjusted to the samevolume. Equal amounts of both soluble and insoluble fractions wereelectrophoretically-separated by SDS-PAGE (Laemmli 1970. Nature 227133-681). The detergent soluble fraction was analysed under bothnon-reducing and reducing conditions while the detergent insolublefraction was examined under reducing conditions. Proteins weretransferred to microporous membranes for immunoblotting (Towbin et al.1979. Proc. Natl. Acad Sci USA 76 4350-4354). Parallel strips weretested with Mab BC4; tissue culture supernatant was used as a negativecontrol.

[0079] The negative control blot was blank.

[0080] Mab BC4 detects a protein of 200-210 kDa in non-reduced, but notin reduced, detergent soluble extracts of cervical squamous epithelium.No product was detected in detergent insoluble fraction of cervicalsquamous epithelium.

[0081] Cervical Biopsies

[0082] Most of the data herein has been obtained from extensiveinvestigations undertaken on biopsy material, for the following reasons:

[0083] (a) A single biopsy specimen provides several serial tissuesections (each 5 um thick) for microscopic examination. All sections aretherefore almost identical, and the various cell populations located inthe same positions relative to each other. Similarity in anatomicalstructure, at the cellular level, is therefore maintained betweensamples.

[0084] This enables reactivities of different antibodies to beinvestigated and compared, on the same cell populations, betweensamples.

[0085] Different cell populations within a tissue are identified bytheir individual morphologies and locations. Therefore, the reactivitiesof individual antibodies against such cells in a biopsy sample, enablestheir specificities to be determined and established.

[0086] (b) Pathological changes are accompanied by a disruption of thenormal histology of the tissue. Therefore, antibody reactivities canonly be effectively investigated (and correlated with pathologicalconditions) in samples, where the tissue architecture is representativeof the situation in vivo.

[0087] Pathological changes that precede malignancy are classifiedaccording to an established system of grading: CIN I, II and III. Theprogression of disease leads to carcinoma in situ and finally a franktumour.

[0088] (c) Most importantly, the use of biopsy material was necessary todetermine whether modulation of expression of the antibody targetsoccurred as a function of disease in the cervix. The profiles ofantibody reactivities on tissue sections of normal biopsies provided aframework to establish their corresponding reactivities on abnormalsmears. Such reactivities on normal and abnormal biopsies enabled theselection of antibodies for use in a cervical smear screening system, inaccordance with the present invention.

[0089] Cervical Smears

[0090] The cells in a smear sample do not maintain their topographicalrelationships to each other, as in a tissue section of a biopsy.Antibodies selected on the basis of their specificities against variouscell populations (as above), were investigated for their ability toprovide qualitative or quantitative information on normal andpathological smear samples. Their absolute or relative numbers,enumerated with these antibodies, provides a means of determiningdetectable changes in these cell populations.

[0091] Tissue Distribution

[0092] Although the monoclonal antibodies were raised against cervicalepithelium, they would be expected to react against their targetepitopes if present on non-cervical epithelium of shared embryologicalorigin. Therefore, their tissue distribution in other epithelial tissuewas determined, enabling further characterisation at that level.

[0093] Biochemical Data

[0094] These have been mainly derived using extracts of cervical amnionand placental tissue, electrophoretically separated on SDS-PAGE gelsunder reducing or non-reducing conditions. Western blots of the resolvedcomponents were then probed with the panel of monoclonal antibodies,enabling their individual target epitopes to be defined at the molecularlevel.

[0095] The detection of such resolved components under definedexperimental conditions provides information on the molecular structureof the target antigen. If detected under both reducing and non-reducingconditions, it can be deduced to be a non-conformationally dependentmonomeric structure. On the other hand, if only detected undernon-reducing conditions, the epitope is present on a conformationallydependent structure.

[0096] The methods of extraction enable the identified material to beassigned to known classes of cell markers. For example, use ofdetergents do not provide extracts with appreciable amounts ofcytokeratins. The cytokeratins are a complex family of cytoplasmicfilamentous protein structures that are well documented in thescientific literature. They are biochemically and antigenically relatedto varying degrees, and are expressed in different epithelia indifferent combinations of polypeptides. A given epithelium or epithelialcell can therefore be characterized by the specific pattern of itscytokeratin components [Ref: Moll R et al, 1982].

[0097] Furthermore, with the exception of Mab 9G5, the molecular weightsof the target antigens precluded them from belonging to the cytokeratinfamily. A monoclonal antibody (AH3) with pan-Cytokeratin reactivity wasused to confirm that the 9G5 epitope was a non-cytokeratin cell marker.This antibody has been previously described [Ref: Houlihan 1993].

[0098] Where sufficient or suitable biopsy material was not obtainable,established carcinoma cell lines derived from cervical tissue wereutilised to prepare such extracts.

[0099] Trypsin Sensitivity

[0100] The susceptibility of target epitopes to proteolysis by trypsinwas determined. The enzyme's pronounced substrate specificity(restricted to lysine and arginine residues) provides a means ofcharacterising the target epitopes further.

[0101] Materials and Methods

[0102] Standard buffers and reagents were prepared in accordance withestablished procedures that are well documented in the scientificliterature so need not be detailed here. Unless otherwise stated, allchemicals were obtained from Sigma Chemical Co, Poole, Dorset, UK.

[0103] Preparation of Immunogen and Immunisation

[0104] Three sources of cervical cells: (A) smear samples, (B)hysterectomies and (C) premalignant CIN biopsies were evaluated fortheir potential use as immunogen:

[0105] (A)

[0106] Cervical smears from routine samples were dispensed into sterilePBS and washed twice. The yield, composition and viability of cells insmears taken from different sites within the cervix was examined. Thecellular composition of conventional smears taken from the exocervixusing a spatula, was compared with endocervical smears taken with abrush. Yields of squamous cells in exocervical smears varied; obtainingup to 10⁶, with viabilities of 50-60%. Endocervical smears gave muchlower yields of typically 10⁴, which also contained predominantlysquamous cells.

[0107] Cytospin preparations of cervical smear samples were alsoexamined immunohistologically and identified using anti-cytokeratinantibodies. The overwhelming majority were squames; the preparationscontained few basal, parabasal, or columnar epithelial cells. It wasconcluded that cervical smears did not represent an effective source ofmaterial for use as immunogen, since they did not contain a sufficientcross section of cervical epithelial cell populations.

[0108] (B)

[0109] Total hysterectomy specimens are typically obtained from womenwith menorrhagia or fibroids; in these, the cervix is essentiallynormal. Such specimens are typically in the perimenopausal (38-45 yrold) period. The optimal method for obtaining single cell suspensionsfrom whole normal cervical biopsy material was determined to be asfollows.

[0110] The excess stromal tissue was removed with a scalpel. Thefragments of tissue were floated in a solution of the enzyme Dispase II(1.2 units/ml, Boehringer Mannheim, Sussex, UK) in HANKS buffered salinelacking Ca²⁺/Mg²⁺ overnight at 4° C. This procedure disrupted theepithelial/stromal junction, such that sheets of epithelial cells couldbe gently teased apart. They were washed by low speed centrifugation inHANKS buffered saline lacking Ca²⁺/Mg²⁺ and resuspended in 0.05%trypsin/0.02% EDTA (both w/v). After a 30 minute incubation at 37° C.with stirring, trypsinization was arrested by the addition of 1.3 mg (in5 ml of saline) of soya bean trypsin inhibitor.

[0111] The secondary enzyme digestion with trypsin disaggregated theepithelial sheets to generate a single cell suspension.

[0112] Large fragments were allowed to settle out under gravity and thesupernatant was removed. The cell suspension was drawn through a 16gauge needle to break up cell clumps, and filtered through a 100 umgauze. Cells were washed twice in PBS prior to use as immunogen.

[0113] (C)

[0114] In addition to normal cervical biopsies, immunogen frompathological CIN II/III biopsies were also used. The material wastreated similarly, except for the initial overnight enzyme treatmentalso being in trypsin.

[0115] The immunisation regime consisted of an initial intra-peritonealinoculation of 5×10⁵ cells as a priming dose, followed by five furtherimmunisations of 0.5-2×10⁶ normal epithelial cells, at 2-3 weeklyintervals in 8 week old female Balb/c mice.

[0116] As some proteolytic cleavage of cell surface components waspossible, the effect of enzyme treatment on the integrity of epithelialcell surface markers was ascertained. This was determined by monitoringits effect on CD44, CD55 and HLA Class 1 antigens, all of which areexpressed on cervical epithelial cells. These established cell surfacemarkers can be detected by appropriate monoclonal antibodies which arereadily commercially available [Ref: Knapp W, 1989]. These markers werefound to be lost or reduced as a consequence of prolonged exposure totrypsin at 37° C. However, adopting measures to minimise such effectsi.e. reduced exposure at 37° C. and use of the enzyme dispase in theinitial overnight incubation at 4° C. instead, ensured that thephenotypic profile of the disaggregated cells was maintained veryclosely to that of native cervical epithelium.

[0117] Fusion and Assay of Supernatants from Hybridomas

[0118] Splenocytes from the immunised mice were fused with NS1 murinemyeloma cells, and hybrids selected by conventional methodology that isdescribed in a number of publications [Ref: Kennet R et al, 1980, andSchrier M et al, 1980].

[0119] Candidate monoclonal antibodies were selected on the basis oftheir reactivities on tissue sections of cervical biopsies usingestablished immunohistological techniques [Ref: Holmes C H et al, 1990].

[0120] Briefly, sections were cut from frozen tissue blocks in acryostat at 5 μm thickness, thawed, air dried at room temperature for 1hour, fixed in ice-cold acetone for 10 mins and immunostained by anindirect immunoperoxidase technique. Supernatants, from wells containinggrowing hybridomas, were incubated for 45 mins at room temperature ontissue sections. After washing in TBS for 5 minutes, they were incubatedfor 30 mins with a commercially available HRPO-conjugated rabbitanti-mouse Ig reagent, optimally diluted in TBS containing 10% normalhuman serum. After two more washes, sections were developed with DAB andhydrogen peroxide for 5 mins. After stopping the reaction by washing theslides in tap water for 5 mins, the sections were counterstained inhaematoxylin, dehydrated, cleared in Histoclear and mounted in DPXmountant.

[0121] Candidate hybridomas, secreting antibodies of interest, werecloned to stability by the method of limiting dilution. The antibodiessecreted by such clones were reassayed to confirm antibody specificityby immunostaining, as above.

[0122] Ig production by hybridomas was also screened by an ELISAtechnique, using commercially available reagents from Dako AS,Copenhagen, Denmark): Rabbit anti-mouse Ig (product no: Z259) optimallydiluted at {fraction (1/2000)} was used as the solid phase capturereagent. Supernatants from hybridomas were incubated for 60 min; boundantibody was detected with a HRPO-conjugated rabbit anti-mouse Ig(product no: P260) diluted {fraction (1/1000)} incubated for 45 mins.Both incubations were at room temperature; the reagents were diluted inPBS, and the wells washed between incubations with PBS-0.025% Tween.

[0123] Biochemical Characterisation

[0124] The methods utilised are generally well known and documented inpublished laboratory technical manuals [Ref: Harlow E and Lane D, 1988and Work and Work, 1979] and in individual publications [Ref:Marchalonis, 1969; Markwell, 1982; and Laemmli, 1970]. Detaileddescriptions of specific procedures undertaken in these investigationshave been previously published [Ref: Holmes C H et al 1990 and HoulihanJ M et al, 1992].

EXAMPLE 1 Mab 6B5

[0125] This antibody of isotype IgG1 was raised against an immunogenprepared from squamous epithelial cells isolated from the cervices ofhysterectomy specimens.

[0126] Reactivity

[0127] Its specificity is mainly against parabasal and basal cells incervical squamous epithelium together with basement membrane. However,it also crossreacts with columnar epithelium and non-epithelial stromalelements.

[0128] Although parabasal cells are not present in large numbers innormal smears, the antibody does identify the small number that arepresent; based cells are usually not present in smear samples. However,6E5 reactivity increases significantly in pathological conditions. Withtumours, 6/7 squamous cell carcinomas showed antibody reactivity. Thisreactivity was retained on one adenocarcinoma; this was expected, as 6B5also reacts with normal columnar cells. There is also an expansion of6B5 reactivity in CINs: in 9/15 moderate to severe CIN specimens tested,there was a marked expansion in the affected squamous epitheliumrelative to the native unaffected epithelium.

[0129] The anti-stromal reactivity is only of relevance in biopsymaterial, as stromal elements are not present in smear samples. Theanti-columnar reactivity is not expected to interfere in the intendedmode of application, as adequate controls exist to correct for thiseffect (see reactivity of Mab 2C7).

[0130] (a) Normal Cervical Epithelium

[0131] No of tests: 90; No of patients: 44 (with TZ: 21)

[0132] Parabasal cells and basement membranes were intensely reactive insquamous epithelium. Columnar cells were also positive, with reactivityexpanded in the transformation zone in 21/21 specimens.

[0133] (b) Premalignant Cervical Epithelium (CIN II/III or III)

[0134] No of tests: 42; No of patients: 30 (with CIN: 15)

[0135] The reactivity was expanded in 9/15 CIN specimens.

[0136] (c) Cervical Carcinomas

[0137] The antibody reacted strongly with 5/7 squamous cell carcinomasand with both adenocarcinomas tested.

[0138] Tissue Distribution

[0139] Despite its restricted distribution in the cervix, the targetepitope is present in other epithelial tissue:

[0140] Placenta

[0141] Epithelial cells in term placental membranes (amnion andcytotrophoblasts) are positive. The basement membrane beneath thesyncytiotrophoblast is positive. In first trimester placenta, bothvillous cytotrophoblast and syncytiotrophoblast are positive. Theantibody shows differential activity with cytotrophoblast inextravillous cell columns: cells at the base of these columns arepositive while those at the periphery are negative. Kidney: glomeruli +;tubules − Pancreas: ducts and acini +; lamina propria − Colon:epithelium +; lamina propria − Liver: hepatocytes +; bileduct/mesenchymal cells − Endometrium: glandular epithelium −; laminapropria −; myometrium −; arteries − Epidermis: parabasal cells +;basement membrane + (foreskin) basal cells +/−; intermediate cells −;superficial cells −

[0142] Biochemical

[0143] Because of its limited reactivity in the normal cervix, anestablished cervical carcinoma cell line C4II was used to characterisethis antibody biochemically. The C4II cell line [Ref: Auersperg N andHauvryl AP, 1962 and Auersperg N, 1969] obtained from ECACC, PortonDown, UK is well documented in the scientific literature. It is alsodeposited in the ATCC under Accession No. CRL 1595.

[0144] Mab 6B5 detects a dimeric product of approximately 181-184 kDa,by immunoprecipitation on the cell surface of C4II. A single componenthaving this molecular weight has subsequently been immunoprecipitateddirectly from radiolabelled membrane preparations of normal invivo-derived cervical squamous epithelial cells. However, the antibodydoes not detect these components on cervical extracts by westernblotting. Taken together, these data indicate that Mab 6B5 detects aconformationally-dependent epitope on a cell surface (non-cytokeratin)protein. The 6B5 target has also been isolated to a high degree ofpurity by immunoaffinity chromatography, for N-terminal sequencing.

[0145] Trypsin Sensitivity

[0146] The reactivity of the antibody is maintained with the C4II cellline after a short 10 min exposure to trypsin at 0.05% (w/v). However,antibody reactivity with placental membranes or amnion cells isabolished after a longer 1 hr exposure to trypsin at 0.1% (w/v). Theantibody target is therefore only partially resistant to trypsin.

EXAMPLE 2 Mab 2C7

[0147] This antibody of isotype IgG1 was raised against an immunogenprepared from normal cervices of hysterectomy specimens.

[0148] Reactivity

[0149] The antibody reacts specifically and solely with columnarepithelial cells.

[0150] (a) Normal Cervical Epithelium

[0151] No of tests: 83; No of patients: 43 (with TZ: 24)

[0152] This antibody reacted specifically with columnar epithelialcells, and not with any other cell population in the cervix. Thesquamous epithelium was negative in all cases.

[0153] (b) Premalignant Cervical Epithelium (CIN II/III or III)

[0154] No of tests: 42; No of patients: 30 (with CIN: 15)

[0155] No reactivity with either normal or neoplastic epithelium; onlyadjacent columnar epithelium was stained.

[0156] (c) Cervical Carcinomas

[0157] The antibody did not react with 7/7 squamous carcinomas tested,as expected. However, it reacted with one of the two adenocarcinomastested.

[0158] It can be concluded that the target epitope of Mab 2C7 is solelyrestricted to columnar cells, and not expressed in either normalsquamous epithelium or in CIN lesions. The antibody is considered usefulsince the presence of columnar cells on a smear indicates that thesquamo-columnar junction has been sampled, and hence that the smear ispatent (or adequate). A correctly sampled smear therefore containsbetween 1-5% of 2C7 reactive cells.

[0159] The 2C7 target epitope is distinct from that of 6B5. In normalsmears, both antibodies identify the columnar cell population. However,whilst 6B5 additionally reacts with parabasal cells, 2C7 only detectscolumnar cells. Therefore, together, they provide a means of enumeratingboth cell populations by analysis of their individual reactivityprofiles. Tissue Distribution Placenta: The amnion and cytotrophoblastin placental membranes, and syncytiotrophoblast were negative in firsttrimester placentae and at term. Pancreas: Epithelium of ducts + Colon:Glandular epithelium + Tonsil: Stratified epithelium − Epidermis:Foreskin − Liver: Hepatocytes− ; bile duct +/− Endometrium: Epithelium −

[0160] Biochemical

[0161] Detergent extracts of endocervical epithelium were prepared byincubating fragments of endocervix in buffer containing CHAPS. OnWestern blots of material separated by SDS-PAGE, the antibody reactedwith unresolved high Mwt components of >400,000 kDA in these extracts.This was further investigated by resolving the extracted material on 1%agarose gels, which are more suitable than SDS-PAGE gels for largermolecules. The antibody reacted with components that preciselycorresponded to a fraction that also stained with periodic acid-Schiff'sbase, but not with Coomassie Blue. This indicated that the targetepitope represented a mucin or a mucin-associated product.

[0162] Trypsin Sensitivity

[0163] The target epitope appears to be trypsin insensitive, as theantibody reacted with cell preparations of cervical tissue which hadbeen trypsinised for periods of up to 1 hr.

EXAMPLE 3 Mabs 9G5 and HG3

[0164] Both antibodies are of isotype IgG1.

[0165] Mab 9G5 was raised against an immunogen prepared from squamousepithelial cells isolated from normal cervices of hysterectomyspecimens.

[0166] Mab HG3 was raised against an immunogen prepared from cervicalbiopsies of pathological CIN material.

[0167] Reactivity

[0168] Both antibodies react primarly with superficial and intermediatesquamous epithelium in the cervix.

[0169] (a) Normal Cervical Epithelium

[0170] No of tests: 83; No of patients: 44 (with TZ: 21 for Mab 9G5 and24 for Mab HG3)

[0171] Both antibodies have similar patterns of reactivity in the normalcervix, with specificities against superficial and intermediate squamousepithelial cells. They are unreactive with basal cells; however, Mab 9G5reacts with parabasal cells in some specimens, where the reactivityfades within the upper parabasal layer. In contrast, Mab HG3 does notreact with parabasal cells in any of the specimens.

[0172] Mab 9G5 does not react with columnar cells; however, Mab HG3shows weak reactivity on columnar cells in a small number of specimens.

[0173] (b) Premalignant Cervical Epithelium (CIN II/III or III )

[0174] No of tests: 42; No of patients: 30 (with CIN: 15)

[0175] In general, both antibodies show detectable differences betweennormal and abnormal cervical epithelium. The reactivity is modulated inCINs, where it is either reduced or absent; e.g. the reactivity of Mab9G5 is markedly reduced in 10/15 specimens. In these lesions, the depthof immunostaining (denoting antibody reactivity) is reduced in terms ofthe number of cell layers, when compared to normal squamous epithelium.

[0176] (c) Cervical Carcinomas

[0177] Both antibodies differed in their reactivities against the sevensquamous cell carcinomas tested:

[0178] Mab 9G5 was completely unreactive with 3/7 specimens tested. Theremaining 4 tumours showed heterogenous reactivity.

[0179] Mab HG3 showed extensive reactivity with 3/7 of the squamous cellcarcinomas, some reactivity with 2, and was unreactive with theremaining 2.

[0180] Heterogeneity is defined as areas of both reactivity andunreactivity within a particular specimen. Generally, despite similarreactivities in the normal cervix, the reactivity of Mab 9G5 with thesetumours was more limited than that of Mab HG3. In particular, Mab 9G5was negative with two tumours which Mab HG3 reacted extensively with.

[0181] When tested on two adenocarcinomas, Mab HG3 reacted with bothtumours; whereas Mab 9G5 was unreactive against both.

[0182] Mabs 9G5 and HG3 recognise distinct target epitopes on the samecell populations. However, it is considered desirable to utilise them intandem, for the following reasons:

[0183] (a) It is not known whether all cells that comprise theintermediate and superficial cell population display both targetepitopes. Even if they normally do, their expression may modulate withstages of the cell or oestrus cycles.

[0184] (b) As superficial epithelial cells are a mainly dead or dyingcell population, it is conceivable that their surface markers would beheterogenous in their detectability.

[0185] (c) Cervical smears are largely comprised of superficial andintermediate cells; with columnar and parabasal cells being a minority.Consequently, a decisive factor in deriving conclusions regarding theclinical status of the sample, would be based on the informationconcerning these cells.

[0186] For these reasons, it may be prudent to avoid dependence on asingle antibody. Therefore, both antibodies may be utilised to enumerateor analyse absolute or relative numbers of intermediate and superficialsquamous cells in normal or pathological conditions. Tissue DistributionKidney, pancreas: Negative liver and endometrium Colon: Mab 9G5 − ; MabHG3 + Tonsil: Stratified epithelium + Epidermis (foreskin): Superficialand intermediate cells + Placenta : Amnion and cytotrophoblast inplacental; membranes + Synciotrophoblast +

[0187] [In the first trimester, these trophoblast populations wereeither negative or only weakly stained.]

[0188] Biochemical

[0189] Mab 9G5 cross-reacts with amnion and placental trophoblastepithelium. On immunoblots, it detects a prominent 40 KDa componentunder both reducing and non-reducing conditions, from detergentextracted material. Therefore, the 9G5 target epitope is likely to be ona non-cytokeratin monomeric protein, which is not conformationdependent. Micro-sequence analysis indicates that the N-terminus isblocked by a methionine residue; further sequence analysis is inprogress on proteolytic digests of the intact molecule. Mab HG3 detectsa 180 KDa component under reducing and non-reducing conditions, in bothdetergent soluble and membrane preparations of both amnion and cervix.Therefore, its target epitope is also likely to be a conformationallyindependent monomeric protein.

EXAMPLE 4 Mab BC4

[0190] This antibody of isotype IgM, was raised against epithelial cellsisolated from premalignant cervical biopsies containing CIN.

[0191] Reactivity

[0192] The antibody primarily reacts with parabasal and intermediatecells in the cervix.

[0193] (a) Normal Cervical Epithelium

[0194] No of tests: 85; No of patients: 44 (with TZ: 24) Typically, thereactivity encompasses 2-6 cell layers above the parabasal layer, andtherefore can include the lower layers of intermediate squamous cells.Basal cells, superficial squames and columnar cells are negative.

[0195] This reactivity pattern, when compared with that of Mabs 6B5, 9G5and HG3 (see Table 1), enables the intermediate squamous cell populationto be enumerated by appropriate deduction.

[0196] (b) Premalignant Cervical Epithelium (CIN II/III or III)

[0197] No of tests: 42; No of patients: 30 (with CIN: 15)

[0198] Reactivity on parabasal cells in CIN specimens is either absentor grossly disrupted in 7/15 specimens. Where there was a complete lossof reactivity, the loss occurred abruptly at the junction between normaland premalignant tissue. Where the reactivity was reduced, it was interms of the number of immuno-stained cell layers.

[0199] (c) Cervical Carcinoma

[0200] The antibody reacted with 2/7 squamous cell carcinomas tested; ofthese one was heterogenous in its reactivity.

[0201] The two adenocarcinomas were unreactive.

[0202] Tissue Distribution Placenta: All placental tissue, both firsttrimester and term are negative. Kidney: Negative Pancreas: NegativeColon: Negative Liver: Negative Endometrium: Negative Buccal cavity:Parabasal cells and stratified epithelium associated with tonsils arepositive. Epidermis: Parabasal cells are positive. (foreskin)

[0203] Biochemical

[0204] The target epitope was probed on immunoblots fromdetergent-solublised extracts and membrane preparations of enzymedisaggregated cervical epithelial tissue. The antibody detected a 200KDa structure under non-reducing conditions only. The target epitope istherefore likely to be present on a conformationally-dependent,non-cytokeratin, protein.

EXAMPLE 5 Assessment of Cervical Cells; Comparison with PAP Test

[0205] The work described in the preceding examples has resulted in thegeneration of a panel of antibodies with reactivities against epithelialcell populations of the human cervix. The reactivities generallyoverlap, with the exception of Mab 2C7 which reacts specifically withcolumnar cells. The overlapping specificities of the other fourantibodies (6B5, BC4, 9G5 and HG3) demonstrate a continuum ofreactivities that parallel the differentiation lineage of cervicalsquamous epithelial cells themselves. It is most probable that theantibody target epitopes are normal differentiation markers which areexpressed and lost as the cells differentiate from basal to terminalsuperficial squames.

[0206] The data herein suggests a scenario wherein with the onset ofpremalignant (CIN) or neoplastic disease conditions, cells can becomearrested at a particular stage of differentiation. This may result in anexpansion of a particular cell population expressing a detectable cellmarker. For example, the anti-parabasal cell reactivity of Mab 6B5 isexpanded in specimens with severe CIN II/III.

[0207] Similarly, a pathological condition may result in an expandedcell population accompanied by the concomitant loss of a characteristiccell marker. For example, the reactivity of Mab BC4, which also reactsagainst parabasal cells in normal epithelium, is reduced in CIN samples.

[0208] The reactivities of Mabs 9G5 and HG3 against intermediate andsuperficial squames is markedly reduced in premalignant CIN samples.This could be due to a reduction in the absolute number of intermediateand superficial squames, as a consequence of arrested differentiation atthe parabasal stage. Conversely, it could be due to a loss of therelevant cell markers themselves, as a result of the pathologicalcondition.

[0209] It should be noted that an actual expansion or reduction in theabsolute numbers of a particular cell population is not a pre-requisitefor the purposes of the present invention. Any detectable change inantibody binding relative to established parameters of normality isrelevant and of use in the present invention. No particular theory orhypothesis limits the nature and scope of the present invention.

[0210] A data bank of numerical parameters pertaining to normal smearsestablishes the “confines of normality”, against which test samples maybe compared. Any significant variance from the established parametersindicates a need for individual diagnosis by suitably qualifiedpersonnel, to assess the clinical status, ie suspect samples arehighlighted using the present invention for further examination.

[0211] Experimental Results

[0212] Reactivity of the antibodies 9G5, HG3, 6B5, BC4 and 2C7 wasassessed on normal cervical smear samples and CIN-2/CIN-3 cervical smearsamples, which were also analysed by the PAP test. The results are shownin Tables 3 and 4.

[0213] Normal cervical smear material was collected in parallel withpathological smear samples in a colposcopy clinic. Following thedeposition of the primary smear on a glass slide for Papanicolaoustaining, the sampling device, either spatula or brush, was placed in 10mls of Hanks buffered saline and agitated. Samples showing obviouscontamination with blood were discarded. The resulting cell suspensionswere then washed ×2 in this buffer and used to generate cytospins.

[0214] Each cytospin spot contained approximately 10⁴ cells.

[0215] The MAb reactivities were detected by an indirect immunostainingtechnique using a Streptavidin-Biotin, alkaline phosphatase detectionsystem. The chromogen (Fuschin; Dakopatts) produced a red stain. Cellnuclei were counterstained with Mayer's haematoxylin.

[0216] Scoring of % cells stained was determined by counting dispersedcells; total cells stained red/total no. nuclei. Cell clumps are oftenpresent in such preparations and the reactivity of the Mab with these isindicated separately.

[0217] The status of the smear sample, normal or CIN, is given asdesignated by cytological examination (PAP) of the parallel specimen.

[0218] Table 3 shows that a pattern of normality for binding of the fivemonoclonal antibodies can be perceived. In the CIN samples binding ofthe same antibodies deviates from this pattern of normality, as shown inTable 4.

[0219] It should be noted that in the case of specimens 6 and 7,visually these samples appeared similar to normal smears. Onmorphological grounds, the majority of cells were obviously superficialsquames and dyskaryotic cells were not apparent. In other specimens, forexample, 1, 2, 3 and 10, dyskaryotic cells were clearly evident.

[0220] Thus, one can establish a pattern of normality for binding of apanel of antibodies to a sample containing cells of the cervix such thatdeviation from that pattern in binding of those antibodies to a testsample is indicative of some abnormality which warrants furtherinvestigation.

[0221] All documents mentioned herein are incorporated by reference.

[0222] References

[0223] Auersperg N and Hauvryl A P, J Nat Cancer Inst 28: pp 605-627,1962.

[0224] Auersperg N, J Nat Cancer Inst 43: pp 151-173, 1969.

[0225] Harlow E and Lane D (Eds), Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988.

[0226] Holmes C H et al, J Immunology 144:, pp 3099-3105, 1990.

[0227] Houlihan et al, J Immunology 149: pp 668-675, 1992.

[0228] Houlihan J M, PhD Thesis, University of Bristol, 1993.

[0229] Kennet R et al (Eds), Monoclonal Antibodies and T-Cell Hybridomas(Plenum Press, 1980).

[0230] Knapp W (Ed), Leukocyte Typing IV, Academic Press, London, 1989.

[0231] Laemmli U K, Nature 227: pp 133-681.

[0232] Marchalonis J J, Biochem J. 113: pp 299-305.

[0233] Markwell, Analyt. Biochem. 125; pp 427-432.

[0234] Schrier M et al, Hybridoma Techniques, Cold Spring HarbourLaboratory, 1080.

[0235] Work and Work (Eds), Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier, 1979. TABLE 1 SUMMARY OF MONOCLONALANTIBODY REACTIVITIES AGAINST NORMAL CERVICAL EPITHELIAL CELLS IN BIOPSYPROVEN NORMAL CERVICES. Target Cell Monoclonal Antibodies Populations2C7 6B5 BC4 9G5 HG3 Basal − +/− − − − Parabasal − + + +/− − Intermediate− − +/− + + Squames Superficial − − − + + Squames Columnar + + − − −

[0236] REACTIVITY OF MABS 9G3 AND HG3 ON CERVICAL CARCINOMAS Tumour Type2C7 6B5 BC4 9G5 HG3 A Squamous − +++ − +/− − B Squamous − − − − +++ CSquamous − +++ − +/− +++ D Squamous − + − − − E Squamous − + +/− +/− +/−F Squamous − +/− − − +++ G Squamous − − +/− +/− +/− H Adenoc. +++ + − −+++ I Adenoc. − + − − +

[0237] TABLE 3 REACTIVITY OF MONOCLONAL ANTIBODIES WITH NORMAL CERVICALSMEARS. (Squamous) (Parabasal) (Columnar) Specimen 9G5¹ HG3¹ 6B5² BC4²2C7² 1. 75% 88% −ve −ve −ve 2. 68% 86% −ve −ve −ve 3. 65% 70% −ve−ve >50   4. 75% 85% −ve −ve −ve 5. 62% 79% −ve −ve −ve 6. 93% 68% 5 1−ve 7. 83% 74% 2 −ve >100    8. 72% 85% 7 3 +ve^($) 9. 68% 90% −ve −ve10 10. 85% 90% −ve −ve −ve mean 74.6 ± 9.79 81.5 ± 8.22

[0238] TABLE 4 REACTIVITY OF MONOCLONAL ANTIBODIES WITH CERVICAL SMEARSFROM PRE-MALIGNANT SPECIMENS WITH CIN-2/CIN-3 (Squamous) (Parabasal)(Columnar) Patient Grade 9G5¹ HG3¹ 6B5² BC4² 2C7² 1. CIN-3 <5% <2% −ve−ve +ve^($) 2. CIN-3 NT <5% −ve −ve +ve^($) 3. CIN-3 <20%   ND >50^($)ve  10^($) 4. CIN-3 60% 20% −ve vs  1 5. CIN-3 50% 96% −ve −ve −ve 6.CIN-3 75% 73% 10 5 −ve 7. CIN-3 86% 78% ve −ve 10 8. CIN-2/3 54%<10%   >20 2^($) −ve 9. CIN-2/3 59% 52% ve −ve 20 10. CIN-2 15% 15% 19−ve 4% 11. CIN-2 38%^($) 20% vs −ve  1

1. A method of determining abnormality in a tissue sample containingcells of the cervix, the method comprising determining binding ofspecific binding substances to the sample and comparing the binding withthe pattern of binding of said specific binding substances to a normalcervical cell sample.
 2. A method according to claim 1 wherein thespecific binding substances include one or more polypeptides eachcomprising an immunoglobulin antigen binding domain.
 3. A methodaccording to claim 2 wherein the specific binding substances include oneor more polypeptides each comprising the immunoglobulin antigen bindingdomain obtainable from a hybridoma selected from those deposited at theEuropean Collection of Animal Cell Cultures (ECACC), Centre for AppliedMicrobiology & Research, Salisbury, Wiltshire SP4 0JG, United Kingdom onFeb. 6, 1995 under the accession numbers ECACC 95020718, 95020716,95020720, 95020717 and
 95020719. 4. A method according to claim 1 orclaim 2 wherein the specific binding substances include one or more suchsubstances which compete for binding to cervical tissue with one or moreantibodies obtainable from a hybridoma selected from those deposited atthe European Collection of Animal Cell Cultures (ECACC), Centre forApplied Microbiology & Research, Salisbury, Wiltshire SP4 0JG, UnitedKingdom on Feb. 6, 1995 under the accession numbers ECACC 95020718,95020716, 95020720, 95020717 and
 95020719. 5. A hybridoma selected fromthose deposited at the European Collection of Animal Cell Cultures(ECACC), Centre for Applied Microbiology & Research, Salisbury,Wiltshire SP4 0JG, United Kingdom on Feb. 6, 1995 ECACC 95020718,95020716, 95020720, 95020717 and
 95020719. 6. Use of a hybridoma ofclaim 4 and/or immunoglobulin antigen binding domain obtainabletherefrom in the obtention of one or more specific binding substances ofuse in assessment of the state or condition of cervical cells in atissue sample.
 7. A specific binding substance including animmunoglobulin antigen binding domain obtainable from a hybridomaselected from those deposited at the European Collection of Animal CellCultures (ECACC), Centre for Applied Microbiology & Research, Salisbury,Wiltshire SP4 0JG, United Kingdom on Feb. 6, 1995 under the accessionnumbers ECACC 95020718, 95020716, 95020720, 95020717 and
 95020719. 8. Aspecific binding substance which competes for binding to cervical tissuewith a specific binding substance according to claim
 6. 9. Use of aspecific binding substance according to claim 6 or claim 7 forassessment of the nature or condition of cells of the cervix in a tissuesample.